Alkaloids from piper longum protectdopaminergic neurons againstinflammation-mediated damage inducedby intranigral injection of lipopolysaccharideHuan He1, Wei-Wei Guo1, Rong-Rong Xu1, Xiao-Qing Chen1, Nan Zhang1, Xia Wu1* and Xiao-Min Wang2
Abstract
Background: Alkaloids from Piper longum (PLA), extracted from P. longum, have potent anti-inflammatory effects.The aim of this study was to investigate whether PLA could protect dopaminergic neurons against inflammation-mediated damage by inhibiting microglial activation using a lipopolysaccharide (LPS)-induced dopaminergicneuronal damage rat model.
Methods: The animal behaviors of rotational behavior, rotarod test and open-field test were investigated. Thesurvival ratio of dopaminergic neurons and microglial activation were examined. The dopamine (DA) and itsmetabolite were detected by high performance liquid chromatography (HPLC). The effects of PLA on theexpression of interleukin (IL)-6, interleukin (IL)-1β and tumor necrosis factor (TNF)-α were detected by enzyme-linkedimmunosorbent assay (ELISA). Reactive oxygen species (ROS) and nitric oxide (NO) were also estimated.
Results: We showed that the survival ratio of tyrosine hydroxylase-immunoreactive (TH-ir) neurons in the substantianigra pars compacta (SNpc) and DA content in the striatum were reduced after a single intranigral dose of LPS(10 μg) treatment. The survival rate of TH-ir neurons in the SNpc and DA levels in the striatum were significantlyimproved after treatment with PLA for 6 weeks. The over-activated microglial cells were suppressed by PLAtreatment. We also observed that the levels of inflammatory cytokines, including TNF-α, IL-6 and IL-1β weredecreased and the excessive production of ROS and NO were abolished after PLA treatment. Therefore, thebehavioral dysfunctions induced by LPS were improved after PLA treatment.
Conclusion: This study suggests that PLA plays a significant role in protecting dopaminergic neurons againstinflammatory reaction induced damage.
BackgroundParkinson’s disease (PD) is one of the most commonneurodegenerative disease, characterized by slow andprogressive death of dopaminergic neurons in thesubstantia nigra pars compacta (SNpc) [1]. Althoughthe mechanism of neuronal degeneration remains notthroughly elucidated, increasing evidences suggest that
neuroinflammatory processes may be involved in theprogressive death of dopaminergic neurons [2–4].Studies have shown that the activation of microgliaplays a key role in neuroinflammation and a largequantity of reactive microglia was found in the SNpcof PD patient [5, 6]. Under normal circumstances,microglia typically exists in a resting state, which involvedin immune surveillance and host defense against immu-nological stimuli. However, it comes to be activatedwhen various pathogenic stimuli appear such as braininjury [7, 8]. Over-activated microglia produces variousneurotoxic factors, such as nitric oxide (NO), tumor
* Correspondence: [email protected] Key Lab of TCM Collateral Disease Theory Research and School ofTraditional Chinese Medicine, Capital Medical University, 10 Xitoutiao,Youanmen, Beijing 100069, ChinaFull list of author information is available at the end of the article
necrosis factor-alpha (TNF-α), interleukin-1β (IL-1β),prostaglandin E2 (PGE2), and reactive oxygen species(ROS), which lead to neuronal damage and results in aself-amplifying cycle of neuronal death [8, 9].In the past, levodopa is the most widely used drugs for
PD treatment, which mainly focused on dopamine (DA)compensation. But years of levodopa treatment causesmotor complications, dyskinesia and its efficacy is coun-teracted [10]. Therefore, new drugs with novel mechan-ism for the treatment of PD are urgently needed. Severalreports have shown that a number of traditional ChineseMedicine have neurotrophic and neuroprotective pro-perties in PD animal models [11–13].Piper longum L. (Piperaceae) has been used as tra-
ditional medicine in Asia and the Pacific Islands. Piperineis a main compound in P. longum. Alkaloids from Piperlongum (PLA) are extracted from P. longum seed. Itmainly contains 53.08 % piperine and 1.73 % piperlongu-minine. Our previous work revealed that PLA possessneuroprotection function on dopaminergic neuronsagainst 6-OHDA-induced damage and in the MPTPanimal model of PD [14, 15]. Recently, another reportshowed that piperine and piperlonguminine protectrotenone-induced neuronal injury [16]. Besides, ourprevious work also showed that PLA significantly sup-pressed BV2 cells activation, attenuated expression ofcyclooxygenase (COX)-2 and inhibited the excessiveproduction of proinflammatory mediator IL-1β andPGE2 in LPS-induced BV2 cells [17].Based on the previous studies, we hypothesized that
PLA showed neuroprotective effects by reducing inflam-mation. In the present study, we used the classic lipo-polysaccharide (LPS)-induced rat model of PD toexamine whether PLA protects dopaminergic neuronsagainst inflammation-mediated damage by inhibitingmicroglial activation.
MethodsPLA extract and reagentsPiper longum was purchased from Anguo, Hebei province,China, in 2014 and identified by Rong Luo, associateprofessor, School of Traditional Chinese Medicine, CapitalMedical University. The voucher specimens of this mater-ial have been deposited in School of Traditional ChineseMedicine, Capital Medical University.The PLA extract was made as our previous work
described [15]. The content of total alkaloids was 74.6 %determined by UV, meanwhile the contents of piperineand piperlonguminine were 53.08 and 1.73 % respectivelydetermined by HPLC. PLA was analyzed in a previousstudy carried out by our laboratory. Refer to thiswork for information about detailed compositions andchromatogram of PLA [15].
Lipopolysaccharide (LPS, from Escherichia coli. serotypeO26:B6), dopamine (DA), 3,4-dihydroxyphenylacetic acid(DOPAC), mouse anti-tyrosine hydroxylase antibody,TritonX-100 and apomorphine were purchased fromSigma-Aldrich (St. Louis, MO, USA). Rabbit anti-Iba-1antibody was purchased from BOSTER (Wuhan, China).Interleukin-1β (IL-6), interleukin-1β (IL-1β), tumor necro-sis factor-alpha (TNF-α) and reactive oxygen species(ROS) enzyme-linked immunosorbent assay (ELISA)kits were purchased from MultiScience Biotech Co., Ltd(Hangzhou, China). Nitric oxide (NO) kit was purchasedfrom NanjingJiancheng Bioengineering Institute (Jiangsu,China).
Animals and surgeryEighty adult male Sprague-Dawley rats (weight 260–300 g)were purchased from the Beijing Vital River Lab AnimalTechnology Co. Ltd. (Beijing, China) and maintainedunder standard conditions with a standard 12-h on/offlight cycle, with food and water supplied ad libitum.After allowed to acclimate to their new surroundingsfor 1 week before experimental surgery, the rats wereinjected 2.0 μL LPS dissolved (5 mg/mL) in phosphate-buffed saline (PBS) into the right SNpc following a previ-ous described protocol [18]. The injection position wasanteroposterior −5.3 mm, lateral 2.0 mm and dorsoventral7.8 mm from bregma. Sham-operated animals wereinjected 2 μL PBS into the right SNpc. Our reasearchhad acquired the ethics approval by Animal Experimentsand Experimental Animal Welfare Committee of CapitalMedical University and all experimental procedureswere approved by the Committee. The ethics approvalnumber is AEEI-2014-081.
Experiment designThe rats were randomly divided into five groups: thesham-operated group (n = 16), the LPS-injected groupfollowed by vehicle treatment (model group, n = 16), theLPS-injected group followed by treatment with 25, 50and 75 mg/kg PLA, respectively (n = 16 each group).Rats in three PLA treatment groups were intragastricallyadministered with PLA (dissolved in 0.5 % sodiumcarboxymethylcellulose) once a day after the surgeryfor 6 weeks. The sham-operated and model groupsreceived 0.5 % sodium carboxymethylcellulose.
Rotational behavior assayOn the second day after treatment with PLA for 3 and6 weeks, the rats were injected hypodermically with0.5 mg/kg apomorphine dissolved in physiological salineto examine the rotational behavior. The number of turnsperformed over 30-min testing period was counted.
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Rotarod testAll rats underwent a 3-day training program on arotarod before the LPS injection, by which time a steadybaseline level of rotarod performance was attained.Briefly, the rats were placed on the rod and sequentiallytested at the speed accelerated from 0 to 40 rpm within2 min. The time latency to fall from the rotarod ateach speed level was recorded. At 3th and 6th week aftertreatment with PLA the rats were tested respectively.
Open-field testRats in each group were tested by the Tru Scan activitymonitoring system (Coulbourn Instruments), whichcontains acoustic insulation and lucifugal field (60 cm×60 cm× 65 cm). Infrared device was installed at the top ofthe box, which was used to accurately track the movementand the behavior. After the end of each test, 75 % ethanolwas used to thoroughly clean the open-field apparatus.The rat movement was recorded for the following para-meters: total movement distance (cm), total movementtime (s), total rest time (s) and horizontal velocity (cm/s).Each test time was 30 min.
Preparation of tissue samplesFour rats from each group were randomly selected formorphological studies on the second day after the finalbehavioral tests. Decapitating all other rats, then thebilateral substantia nigra (SN) and striatum were rapidlydissected and stored at −80 °C. The SN was used forthe quantification of proinflammatory cytokines, andthe striatum for determination of the content of DA.For the morphological studies, rats were deeply anes-thetized with chloral hydrate, then transcardially per-fused with 200 mL saline followed by 200 mL of 4 %paraformaldehyde in 0.1 M phosphate buffer. Brainswere removed and post-fixed in the same fixative andthen immersed in a 20 % sucrose solution and a 30 %sucrose solution. Coronal section were cut on a freezingmicrotome (Leica, Germany) at a thickness of 40 μm andused for immunohistochemistry as described below.
High performance liquid chromatography (HPLC)The determination of DA and its metabolite 3,4-dihy-droxyphenylacetic acid (DOPAC) was carried out usingHPLC with a Coul Array electrochemical detector(Model 5600A, ESA, USA) equipped with Waters sym-metry shield RP 18 column (150 × 3.9 mm, 5 μm).The mobile phase consisted of 50 mM sodium citrate,8 % methanol, 0.1 mM EDTA · 2Na, 0.2 mM 1-octanesulfonic acid sodium salt and was finally adjustedto pH 4.1. The flow rate was 0.8 mL/min. Striatumtissues from 6 animals of each group were used andperformed as described in previous work [19].
Immunohistochemical staining of TH and Iba-1Eight sections were selected for immunohistochemicalstaining of the tyrosine hydroxylase (TH). The mouseanti-TH antibody was diluted at 1:2000. Adjacentsections were used for detection of microglial markerIba-1. The rabbit anti-Iba-1 antibody was diluted at1:200. Sections were perforated with 0.3 % Triton-X100 and blocked with normal horse serum (1:100 dilu-tion), then were incubated with primary antibodies for24 h at 4 °C. After that, sections were incubated withbiotinylated anti-mouse antibody and biotinylated anti-rabbit antibody (Vector laboratories, Burlingame, CA,USA) respectively for 30 min at 37 °C, followed by avidin-biotin-peroxidase (Vector laboratories, Burlingame, CA,USA) incubation for 30 min at 37 °C. Finally, the immunecomplex was detected by 3, 3’ - diaminobenzidine (DAB).To measure the numbers of TH-ir cells in the SN,
stereological cell counting was performed. The opticalfractionator method on a Stereo Investigator system(Micro Bright Field, USA) was used to count the totalnumbers of TH-ir neurons in the SN and a Leica micro-scope was used. The survival rate of TH-ir neurons inthe SN was determined by counting the number ofTH-ir neurons on LPS-injected side relative to thenumber of TH-ir neurons on the non-injected side.Quantitative analysis of Iba-1-stained immunohisto-chemical images were carried out with an Image-ProPlus 6.0 system and positive results were expressed asaverage optical density value. All sections were codedand examined blindly.
IL-6, IL-1β and TNF-α immunoassayThe right SN was used to detect the proinflammatorycytokines. Tissues were made into 10 % homogenate andthen the homogenate was centrifuged at 3000 g for15 min at 4 °C. The supernatant was collected at 4 °C.IL-6, IL-1β and TNF-α were detected using the commer-cial enzyme-linked immunosorbent assay (ELISA) kits(MultiScience Biotech, Hangzhou, China). All experi-mental procedures were performed according to themanufacturer’s instructions. Besides, we used the BCAprotein kit to measure protein contents in samplesaccording to the manual. The SN tissues from 6 animalsof each group were used.
Measurement of ROS and NOThe SN was also used to measure ROS and NO andtissues were made into 10 % homogenate as above. ROSwas measured using enzyme-linked immunosorbentassay (ELISA) to measure absorbance value at 450 nmwith ELISA kit. The content of NO was measured usingNO assay kit according to the manufacturer’s guidelinesby measuring the absorbance value at wavelength of550 nm. BCA protein kit was used to measure the
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protein contents in samples according to the manual.The SN tissues from 6 animals of each group were used.
Statistical analysisData were processed by commercially available softwareGraphPad Prism 5.0. Results are typically presented asmeans ± S.E.M. Statistical significance was assessed usinga one-way analysis of variance (ANOVA), followed byDunnett post hoc test (compares all the other columnswith the designated control column). Significance wasset at P < 0.05.
ResultsPLA administration improves apomorphine-inducedrotational behaviorThe apomorphine-induced rotational cycles of all groupsare shown in Fig. 1. The rotational cycles of modelgroup animals increased than that of sham group (P <0.001) examined both at 3th and 6th week after LPSinjection. However, treatment with 25, 50 and 75 mg/kgPLA for 6 weeks significantly reduced the numbers of
apomorphine-induced rotational turns compared withthe model group (P < 0.001) (Fig. 1b).
PLA administration improves rotarod behaviorAs apomorphine-induced rotation test, the rotarod testis also a classic method to evaluate behavioral dysfunc-tion of PD model rats. Animals in each group performedequally before LPS injection (Fig. 2a). A significantdecrease of time which rats keep balance on rod wasobserved after LPS injection for 3 and 6 weeks (P <0.001) (Fig. 2). However, treatment with PLA (25, 50 and75 mg/kg) for 6 weeks may significantly improve therotarod behavior (Fig. 2c).
PLA administration improves locomotor activityOpen-field test is a classic behavioral test to comprehen-sively evaluate spontaneous behaviors of rats. The resultsshowed that the most activities in the model group weresignificantly reduced than other groups. As shown inFig. 3a, b, d, the activities of model group such as totalmovement distance, total movement time and horizontalvelocity were all decreased. In contrast, the rest time for
Fig. 1 Effects of PLA treatment on apomorphine-induced rotational behavior. Rats were randomly grouped and then treated with PLA (25, 50 and75 mg/kg) or vehicle for 6 weeks after LPS injection. On day 21 a and 42 b, rats were hypodermically injected with apomorphine (0.5 mg/kg) to inducerotational behavior. The number of turns were recorded for 30 min. n = 10–14. ***P < 0.001 vs. sham group, #P < 0.05, ###P < 0.001 vs. LPS group
Fig. 2 Effects of PLA treatment on rotarod behavior. Rats were randomly grouped and then treated with PLA (25, 50 and 75 mg/kg) or vehiclefor 6 weeks after LPS injection. a A baseline trial was conducted before surgery. b, c The time each group animals remained on the rotarod afterPLA administration for 3 and 6 weeks, respectively. n = 10–13. ***P < 0.001 vs. sham group, ##P < 0.01, ###P < 0.001 vs. LPS group
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Fig. 3 Effects of PLA treatment on locomotor activity. Rats were randomly grouped and then treated with PLA (25, 50 and 75 mg/kg) or vehiclefor 6 weeks after LPS injection. On day 42, rats were placed into open-field apparatus, and spontaneous behaviors of 30 min was measured andanalyzed by a Tru Scan 2.01 software. a total movement distance; b total movement time; c total rest time; d horizontal velocity. n = 10–11.***P < 0.001 vs. sham group, ##P < 0.01, ###P < 0.001 vs. LPS group
Fig. 4 Effects of PLA treatment on the movement track of rats. a sham-operated group; b LPS-injected group followed by vehicle treatment;c the LPS-injected group followed by treatment with 25 mg/kg PLA; d the LPS-injected group followed by treatment with 50 mg/kg PLA;e the LPS-injected group followed by treatment with 75 mg/kg PLA
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Fig. 5 Effects of PLA treatment on the contents of DA and DOPAC in the striatum. Rats were randomly grouped and then treated withPLA (25, 50 and 75 mg/kg) or vehicle for 6 weeks after LPS injection. On day 42, rats were decapitated and the contents of DA andDOPAC in the striatum were detected by HPLC. The survival ratios (right relative to left side) of DA a and DOPAC b were calculated.n = 6. ***P < 0.001 vs. sham group, #P < 0.05, ##P < 0.01 vs. LPS group
Fig. 6 Morphological evidence of the protective effect of PLA against LPS-induced damage to dopaminergic neurons in the SN. Rats wererandomly grouped and then treated with PLA (25, 50 and 75 mg/kg) or vehicle for 6 weeks after LPS injection. On day 42, rats weredeeply anesthetized and transcardially perfused with 4 % paraformaldehyde and processed as above. Frozen sections at a thickness of40 μm were cut and TH was detected by immunohistochemical staining to show dopaminergic neurons in the SNpc. (A1–A4) sham-operatedgroup, TH staining was similar on the non-injected (A2) and injected (A3) side; (B1–B4) model group, LPS-injected followed by vehicle treatment,an obvious loss of TH-ir cells was seen on the injected side (B3); (C1–C4, D1–D4, and E1–E4) the LPS-injected group followed by treatment with 25,50 and 75 mg/kg PLA respectively, much more TH-ir neurons survived on the LPS-injected side compared to model group
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the rats in the model group was significantly higher thanthe others (Fig. 3c). Treatment with PLA (25, 50 and75 mg/kg) for 6 weeks significantly increased the totalmovement distance, total movement time and horizontalvelocity and decreased the rest time. Figure 4 shows theeffect of PLA on the movement track of 30 min of ratsin each group.
PLA administration attenuates depletion of DA andDOPAC in the striatum induced by LPS intranigralinjectionThe DA content in striatum was decreased after injec-tion of LPS in the SN. In the model group, the survival
levels of DA and DOPAC on the LPS-injected side weremuch lower than that of sham group (P < 0.001). Aftertreatment with PLA 50 or 75 mg/kg for 6 weeks, the DAand DOPAC depletion in the striatum induced by LPSintranigral injection was significantly attenuated (Fig. 5).
PLA treatment protects dopaminergic neurons from theinjury induced by LPS intranigral injectionRepresentative microphotographs of TH immunohisto-chemical staining in the SNpc are shown in Fig. 6. Thenumbers of TH-ir neurons on the injection side and non-injection side were similar in sham-operated animals.Model group rats showed a marked loss of TH-ir neuronsand their dendrites on the injection side. However,PLA (25, 50 and 75 mg/kg) treatment significantly recov-ered this loss of nigral TH immunoreactivity. The survivalratio of dopaminergic neuron on the LPS-injected siderelative to the non-injected side was shown in Fig. 7.Compared with the sham group, the survival of TH-irneurons in the SNpc of model group was significantly de-creased (P < 0.001). In contrast, the rats treated with 50and 75 mg/kg PLA after LPS injection showed a great in-crease in the survival rate of TH-ir neurons in the SNpcwhen compared to model group rats (P < 0.01). A lowdose (25 mg/kg) of PLA showed an obvious trend towardincreasing the survival of TH-ir neuros in the SNpc, al-though not statistically significant.
PLA treatment inhibits microglial activation in the SNpcinduced by LPS intranigral injectionImmunocytochemical staining of ionized calcium bindingadaptor molecule-1 (Iba-1) antibody was used to revealmicroglial activation. As shown in Fig. 8, microgliaunderwent a morphological change from resting state
Fig. 7 Effects of PLA treatment on the survival ratio of dopaminergicneurons in the SNpc. The numbers of TH-ir neurons in the SNpc werecounted as described in methods. Survival ratio of the TH-ir neurons inthe SNpc (the injected side relative to the non-injected side) wascalculated. n = 4. ***P < 0.001 vs. sham group, ##P < 0.01 vs. LPS group
Fig. 8 PLA treatment inhibits microglial activation. The morphological changes of microglia in the SNpc were revealed by Iba-1 immunostaining.Representative micrographs were shown. Scale bar represents 100 μm
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to activated cells with larger cell body and branchingcoarsening in the SNpc after LPS injection. However,the activation of microglia was significantly suppressedafter PLA treatment. The average optical density valuewas shown in Fig. 9. Compared with the sham group, theIba-1 content increased significantly in the LPS injectiongroup (P < 0.001), but which was significantly decreasedafter treatment with PLA for 6 weeks (P < 0.001).
PLA treatment inhibits the release of TNF-α, IL-6 andIL-1β in the SN induced by LPS intranigral injectionIntranigral injection of LPS induced excessive releaseof proinflammatory cytokines. The concentrations ofproinflammatory cytokines TNF-α, IL-6 and IL-1β in theSN of model group rats were significantly increased whencompared with the sham-operated group rats (P < 0.01).Treatment with PLA 50 and 75 mg/kg for 6 weeks
significantly decreased the concentrations of TNF-α, IL-6and IL-1β when compared with model group (P < 0.05).Rats treated with 25 mg/kg PLA, only the IL-6 concen-tration was decreased significantly compared with modelgroup (P < 0.05) (Fig. 10).
PLA treatment inhibits the release of ROS and NO in theSN induced by LPS intranigral injectionTo demonstrate if PLA protecting the dopaminergic neu-rons is related to inhibiting the release of ROS and NO,the concentrations of ROS and NO in the SN were ob-served. The data showed that the concentrations of ROSand NO in model group rats were markedly increasedcompared with sham-operated group rats (P < 0.01). Theconcentrations of ROS and NO of rats treated with 50and 75 mg/kg PLA for 6 weeks were fewer than that ofrats in model group (P < 0.05). Rats treated with 25 mg/kgPLA, only the NO concentration showed a significantdecrease compared with model group (P < 0.05) (Fig. 11).
DiscussionA number of researches have suggested that inflamma-tory process plays a significant role in the progressionof PD, especially microglial activation [8, 9, 20]. LPS,a component of the cell wall of Gram-negative bacterial,is extensively used to induce inflammation by activateglial cell [18, 19]. The PD models induced by LPSboth in vitro and in vivo are widely used, which cannot only reflect the role of neuroinflammation in PD butalso have been used in drug discovery [21–23]. Forexample, pioglitazone and naloxone have been studiedfor their potential neuroprotective effects on LPS-inducedPD models [24, 25]. It was reported that, the inflammatoryreaction induced by injecting LPS into SN had a selectiveirreversible and long lasting damage on the dopaminergicneurons, which ultimately resulted in neurodegeneration[20, 22, 26]. Many studies also showed that a single
Fig. 10 Effects of PLA treatment on the concentrations of proinflammatory cytokines in the SN. Rats were randomly grouped and thentreated with PLA (25, 50 and 75 mg/kg) or vehicle for 6 weeks after LPS injection. On day 42, rats were decapitated and the SN wasused to detect the concentrations of TNF-α a IL-6 b and IL-1β c by commercial ELISA kit. n = 6. **P < 0.01, ***P < 0.001 vs. sham group,#P < 0.05, ##P < 0.01 vs. LPS group
Fig. 9 Effects of PLA treatment on the average optical density ofIba-1 in the SNpc. n = 4. ***P < 0.001 vs. sham group, ###P < 0.001vs. LPS group
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intranigral injection of LPS led to depletion of DA andloss of dopaminergic neurons in animals [21, 27, 28].In the present study, the effect of PLA on LPS
induced rat model was examined and three differentbehavioral tests were conducted. The apomorphine-in-duced rotational test was used to estimate the injury de-gree of dopaminergic system [29]. Apomorphine-inducedrotation cycles signifcantly increased in the model grouprats induced by intranigral injection of LPS, in con-trast, PLA treatment showed an improvement effecton this behavioral dysfunction (Fig. 1). The other twobehavioral dysfunctions were also significantly im-proved by PLA treatment measured by rotarod testand open-field test. Further studies suggested thatPLA treatment was able to block the loss of TH-irneurons in the SNpc in LPS intranigral injection rats.Studies have shown that microglial cells are readily
activated and a variety of neurotoxic factors exist inthe SN of LPS-induced PD model rats, which cause thedamage of dopaminergic neurons [30–33]. The neurotoxicfactors include IL-6, IL-1β, TNF-α, O2
−, NO and so on[33, 34]. Our previous work suggested that PLA couldsignificantly inhibit LPS-induced BV2cell activationand proinflammatory mediator production, such asIL-1β [17]. In the present study, our results showedthat an intranigral injection of LPS led the microglialover-activation and the production of a large amountof neurotoxic proinflammatory factors (TNF-α, IL-6and IL-1β), which triggered the cascade of events andcaused the death of neighboring dopaminergic neurons,in contrast, PLA treatment was able to suppress theactivation of microglia and block the release of theseproinflammatory cytokines, attenuate neuroinflamma-tion, and protect dopaminergic neurons damage. Givenour previous work that PLA could cross the blood-brainbarrier [35], these data indicates that PLA has potentanti-inflammatory activity in the central nervous system.Nuclear factor κB (NF-κB) signaling pathway is the most
significant pathway which mediates the LPS-induced
microglial inflammatory response and regulates the pro-duction of various inflammatory mediators [29, 30]. Inour previous work, the role of PLA in modulating NF-κBpathway in BV2 cells was studied [17]. PLA could inhibitthe nuclear translocation of p65 subunit of NF-κB. Thep65 protein level in the nucleus was increased after LPSstimulation, however PLA treatment significantly counter-acted it. What’s more, PLA could also inhibit NF-κBactivity through the inhibitory effect on the degradation ofIκB. Thus, the inhibitory effect on NF-κB activitationmight be involved in the anti-inflammatory propertiesof PLA.In microglial activation, ROS production occurs prior
to the cytokine production and NO is another neurotoxicfactor [33, 36]. A recent study showed that alkaloidsfrom P. longum decreased ROS production, stabilizedmitochondrial membrane potential and inhibited theopening of the mitochondrial permeability transitionpore (mPTP) [16]. In this study, we further investi-gated the effects of PLA on production of neurotoxicfactors ROS and NO induced by LPS intranigral injection.Our results indicated that PLA could reduce the produc-tion of ROS and NO induced by LPS, which might be asignificant mediator of the neuroprotective effect of PLA.Taking our previous work together, PLA might be amulti-component and multi-target approach in thetreatment of PD. Further studies are needed to betterunderstand the mechanism involved in PD treatment.
ConclusionIn this study, the treatment of PLA, an active extractof the traditional Chinese medicine P. longum, was ableto attenuate the depletion of DA and DOPAC in thestriatum, facilitate the survival of damaged neurons byinhibiting microglial activation and suppressing the releaseof neurotoxic factors such as TNF-α, IL-1β, IL-6, ROS andNO, and improve the LPS-induced behavioral dysfunc-tions. In summary, this study suggests that PLA may
Fig. 11 Effects of PLA treatment on the concentrations of ROS and NO in the SN. Rats were randomly grouped and then treated withPLA (25, 50 and 75 mg/kg) or vehicle for 6 weeks after LPS injection. On day 42, rats were decapitated and the SN was used to detectthe concentration of ROS a by commercial ELISA kit and the concentration of NO b by NO assay kit. n = 6. **P < 0.01, ***P < 0.001 vs.sham group, #P < 0.05, ##P < 0.01 vs. LPS group
He et al. BMC Complementary and Alternative Medicine (2016) 16:412 Page 9 of 11
have a protective effect on dopaminergic neurons againstinflammatory reaction induced damage.
AcknowledgmentsThis work was supported by National Natural Science Foundation ofChina (No.81473333) and Capital Research of Traditional ChineseMedicine (NO. 14ZY02).
Availability of data and materialsThe dataset supporting the conclusions of this paper are included withinthe article.
Authors’ contributionsHH carried out the experiments from animal surgery to determinationand finished the manuscript. XW analyzed the feasibility of this studyand revised the manuscript. XMW provided experimental instrumentsand theoretical guidance. WWG and RRX participated in animal surgeryand tissue preparation. XQC and NZ helped designed the experimentand provided suggestions. All authors have read and approved thefinal manuscript.
Competing interestsThe authors declare that they have no competing interests.
Consent for publicationNot applicable.
Ethics approvalThis study was approved by Animal Experiments and ExperimentalAnimal Welfare Committee of Capital Medical University. The ethicsapproval number is AEEI-2014-081.
Author details1Beijing Key Lab of TCM Collateral Disease Theory Research and School ofTraditional Chinese Medicine, Capital Medical University, 10 Xitoutiao,Youanmen, Beijing 100069, China. 2Beijing Institute for Brain Disorders andKey laboratory of Neurodegenerative Diseases of the Ministry of Education,Capital Medical University, 10 Xitoutiao, Youanmen, Beijing 10069, China.
Received: 27 January 2016 Accepted: 13 October 2016
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He et al. BMC Complementary and Alternative Medicine (2016) 16:412 Page 11 of 11
